Polymeric Composition, Material Produced and Their Applications

ABSTRACT

Polymeric composition, material produced thereof and their applications The invention relates to a novel polymer composition comprising at least one copolymer constituted of acrylic (AC) monomer units and of at least one water-soluble comonomer unit, which is preferably 2-acrylamido-2-methyl-propane sulfonic acid (AMPS). The composition is used to produce materials, such as membranes, film or suface coatings, that are suitable as highly tissue compatible supports for epithelial cells, such as hepatocytes. The materials are suitable to be used in a bioartificial liver, where they work as separating membrane and carrier for adhesion-dependent tissue cells. As an example it is described how selectively permeable membranes are formed by a phase inversion process from these acrylic copolymers with varying content of 2-acrylamido-2-methyl-propane sulfonic acid (AMPS) and how they interact with liver cells to construct a biohybrid organ for liver replacement.

The invention relates to a novel polymeric composition, a method for itsproduction, and its use for the production of materials to be employedin medical or biological applications, particularly for biohybrid orbioartificial organs, involving a direct contact of the material withfluids and/or cells and/or tissues.

Biohybrid or bioartificial organs are designed to support or temporarilyreplace human organs with malfunctions. The hybrid usually comprisesmembranes having “passive” separation, filter and carrier functions andalso living organ cells having “active” transport and secretionfunctions.

In artificial organs like the artificial kidney so far only the passiveseparation and filter function of the organ is replaced with the help ofspecialised polymeric membranes. However, in the healthy kidney thefiltration process is followed by an active re-adsorption processrealised by the kidney epithelium. In this process, ingredients of theprimary urine and water are transported against a concentration gradientfrom the primary urine back to the blood. Furthermore the kidneyepithelium possesses an endocrine function responsible for release oferythropoietin, vitamin D hormones, angiotensins II, kinins and reninscontributing to the homeostasis of the organism.

Analogue situations exist for the liver being one of the most importantendocrine gland of the organism secreting hormones and other proteins(albumins, α- and β-globulins, fibrinogen, prothrombin, clottingfactors) with storage function for glycogen, proteins, vitamins, besideits detoxifying function. It is clear that with the help of poremembranes only passive filtration and sorptive functions of this organresponsible for detoxification can be achieved and used for therapeuticpurposes.

Therefore the immobilisation of organ cells in so-called biohybridorgans was proposed to use the active transport and secretion functionof viable cells for supporting or replacing the functions of diseasedorgans. Until now the achieved results are of little therapeutic valuebecause on the one hand the viability of cells can not be maintained forthe necessary time period and on the other hand the functionality of theimmobilised cells achieved so far does hot correspond to thefunctionality of the natural organ.

One of the main reasons of this unsatisfactory functionality originatesfrom the membrane in terms of being a carrier of anchorage dependentcells. Corresponding to the present knowledge the membrane carriershould possess the following properties for optimal functionality of thecells;

1. In general there are insufficient number of primary cells from thepatient's own organ available. Therefore donor cells of human or animalorigin have to be used in biohybrid organs. In every case a shielding ofthe patient from allogenic or xenogenic cells in the bioreactor isnecessary to prevent activation of the host immune system. This immune pisolation limits the maximum pore size of the membrane. Antibodies,complement factors, viruses etc. should not pass through the membrane.On the other hand the permeability of the membrane should permit theexchange of substances necessary for supply to the cells (nutrients,some proteins, such as albumin, gases, toxins to be removed) and removalof the products of the cells (cell metabolites, specific proteins,etc.).

2. Organ cells like hepatocytes are adhesion-dependent and require anextracellular matrix at the contact sites to the membrane where they areanchored. Only in this way do they maintain viable over a longer timeperiod, develop their normal morphology and function and organise inpolar, tissue-like structures. Hence, the membrane must allow themoderate adsorption of extracellular matrix proteins without majorconformational changes and allow their subsequent structuralorganisation. Consequently, the membrane surface contacting the tissuecells has to fulfil extreme requirements to be tissue compatible.

Depending on the organ to be supported or replaced and the requirementsstated in paragraph 1 and 2 the protein adsorption capacity of themembrane has a crucial importance. Endocrine organs produce proteinsthat have to be delivered into the blood stream without major adsorptionduring passage through the membranes to fulfil their function in thehuman body. Thus, the membrane may have only a moderate adsorptioncapacity for proteins.

It is therefore an object of the present invention to provide acomposition and a material made of the composition, both suitable forbeing used in applications involving direct contact with cells,especially tissue cells. The material made from the composition shallsupport moderate adsorption of the cells and support their naturalfunctions. Moreover, the composition should be suitable for preparingmembranes with pore sizes that enable exchange of substances, which arenecessary for the supply of the cells. At the same time, the pore sizeshould ensure an efficient immune isolation to shield a patient from theallogeneic or xenogeneic cells attached to the membrane.

This and other objects are accomplished by a polymeric compositioncomprising at least one acrylic copolymer constituted of 90 to 99 mole-%of acrylic (AC) monomer units and of at least one water-solublecomonomer unit.

Furthermore this and other objects are accomplished by a polymericcomposition comprising at least one acrylonitrile copolymer constitutedof 90 to 99 mole-% of acrylonitrile (AN) monomer units and of at leastone Water-soluble comonomer unit.

The comonomer is advantageously an ionic monomer, particularly ananionic monomer. According to a highly preferred embodiment2-acrylamido-2-methyl-propane sulfonic acid (AMPS) is used as comonomer.Molar contents of AMPS in the copolymer of 1 to 10 mole-%, particularlyof 3 to 5 mole-%, have been shown to be favourable with respect to thedesired product characteristics as outlined above.

The polymeric composition according to the present invention shows highbiocompatibility for many cells, including tissue cells, particularlyepithelial cells. Therefore, the composition is suitable for theproduction of biomaterials, such as membranes, films or coatings, whichcan be used in medical or biological applications involving directcontact of the material with body fluids and/or cells and/or tissues,particularly with hepatocytes. Cells that are immobilised at thematerial can easily be fed by a fluid stream, which supplies the cellswith nutrients thereby enabling exchange of substances into the cellsand outside the cells.

It has been shown that materials, produced of the composition canadvantageously be used in biohybrid or bioartificial organs, where theywork as selectively permeable separating membrane and carrier foradhesion-dependent tissue cells, such as liver, pancreas, lung cells. Asshown by experiments on liver cells immobilised on membranes accordingto the invention, these cells maintain a high degree of naturalcytochrome dependent testosterone and ethoxyresorufin metabolism andalso express glutathione-S-transferase.

Due to their poor cytotoxicity and low affinity for protein adsorption,materials made up of the polymeric composition can also be used formedical applications within the body. For example they may be appliedfor implants or biosensors.

The composition can easily be produced by radical copolymerisation ofAC, particularly of AC, and the at least one water-soluble comonomer inpresence of a suitable radical initiator, such as ammoniumperoxodisulfate, all dissolved in an aprotic solvent, such asdimethylformamide. To obtain the appropriate product characteristics thereaction is preferably carried out at a total concentration of monomersin the reaction solution in the range of 1 to 5 mole/l, particularly ofabout 4 mole/l, and a concentration of the radical initiator of about0,4 to 4 mmole/l.

Membranes, preferably having a rate of water flux through the membranein the range of 1 to 10 l/m²hkPa, particularly of about 2 l/m²hkPa, anda cut-off in the range of 150 to 1,000 kDa, particularly of 200 to 600kDa, even more particularly of 300 to 400 kDa are favourably obtained bya phase-inversion process from the copolymers according to theinvention. In detail, a casting solution, which contains a polymericcomposition at a solid content of for example 15 to 25weight-% dissolvedin a suitable solvent, is prepared. Then the solution is casted on asupport or extruded through a suitable nozzle, particularly of theso-called tube-in-orifice type; and the cast or the extruded solution iscoagulated by a wet or wet-dry-wet process in a coagulation bath to forman asymmetric membrane. Preferably, the membrane is subsequentlysubjected to a wet post-treatment in water or steam.

Other preferred embodiments of the invention are disclosed in thedependent claims.

The invention is described in more detail by way of examples and thefollowing figures:

FIG. 1 Cytotoxicity test of membranes prepared according to examples 3and 4,

FIG. 2 MTT test of 3T3 fibroblast growth on membranes according toexamples 3 and 4 (control—MC).

EXAMPLE 1 Polymer Synthesis

The copolymers according to the present invention generally comprisepoly-acrylonitrile (PAN) derivatives functionalised by the anioniccomonomer 2-acrylamido-2-methyl-propane sulfonic acid (AMPS).

The introduction of the functionality was achieved by means offree-radical random copolymerisation techniques to yield copolymershaving well adjusted composition ratios and good membrane formingproperties. Homopolymerised PAN was included in the studies as referencematerial.

Before use, AN basic monomer (MERCK, >99% purity) was inert fractionaldistilled. AMPS comonomer (ALDRICH, 98% pur.) was applied withoutfurther purification. Dimethylformamide (DMF, ALDRICH, 99,8% pur.) wasinert vacuum distilled at 40 mbar/b.p.60° C. before application asaprotic solvent in polymer synthesis. Free radical initiators2,2′-azoisobutyronitrile (AIBN, recrystallised twice from water at 30°C.) or ammonium peroxidisulfate (APS, fractional crystallised fromethanol at 40° C.) were used in home- and copolymerisations,respectively.

Solution polymerisation initiated by free radicals was carried out as anupscaling procedure (5 l batch size) using a conventional five-neckeddouble-walled jacket glass reactor with an outer thermostatting. Fromthe recipe ingredients, first the main portion of aprotic DMF solventwas filled into the reactor and heated up to the desired temperatureunder continuous stirring (150 r.p.m.) in an inert atmosphere.Subsequently, AN and the comonomer and finally the initiator, dissolvedin the remaining DMF, were added into the batch. The reaction time wasindividually regulated from 360 min (PAN) to 1800 min (AMPS) to obtainthe required conversions in the case of APS initiated copolymer series,the polymerisation temperature was adjusted to 45° C., whereas in thecase of AIBN initiated AN homopolymerisation the temperature was 54° C.as found by kinetic calculations for adjusting the home- and copolymermolecular weights.

Because of initiator differentiation it can be assumed definitely thatthe PAN reference obtained with AIBN is free of any functionalities,even in initiator terminal position.

The applied total monomer concentration of [M]=3,8 mole/l for PAN and5,0 mole/l for AMPS copolymer includes well graduated molar ratios ofthe comonomer in the feed, ranging from zero for PAN reference up to 5,0mole-% for the AMPS enriched feed.

The total monomer concentrations and initiator concentrations of 8mmole/l (AIBN for PAN) or 4 mmole/l (APS for copolymer series) werebalanced in order to obtain number average molecular weights higher than30,000 g/mole for obtaining membrane forming polymers suitable for therequired ultrafiltration pore structure.

After the end of reaction, polymer solutions were poured into a largeexcess of ethanol used as precipitant. The precipitated polymer wasfiltered, manifold washed and rinsed with dehydrated ethanol, which waspredried on air before exhaustive vacuum drying, until constant weightwas reached.

EXAMPLE 2 Polymer Characteristics

The polymers synthesised from AN and AMPS according to example 1 werecharacterised for copolymer composition by elementary analysis andnumber average molecular weight by membrane osmometry. PAN was used asreference material. The results of this analysis are shown in Table 1.TABLE 1 Polymer characteristics. X_(AMPS, Pol.) = comonomerconcentration in copolymer from N-elementary analysis, M_(n,Osm.) =molecular weight of copolymer measured in 0.1 n NaNO₃/DMF-electrolytesolution. Polymer characteristics Polymerisation X_(AMPS,Pol.)M_(n,Osm.) pattern Abbreviation [mole %] [g/mole × 10⁻³] PAN PAN — 48.5P(AN/AMPS)1/1 AMPS1/1 2.9 33.5 P(AN/AMPS)1/2 AMPS1/2 3.2 32.3P(AN/AMPS)1/3 AMPS1/3 2.9 45.3 P(AN/AMPS)2/1 AMPS2/1 5.2 57.3

EXAMPLE 3 Membrane Formation

Membranes were formed by a phase inversion process from a solution ofthe polymers according to examples 1 and 2 in dimethylformamide. Thepolymer concentration varied from 10 to 22,5 wt-%. The solution was caston a non-woven support (AMPS1/1, 1/2 and 2/1) or directly on a stainlesssteel band with a casting slit of 300 μm and a drawing speed of 2 to 4m/min. After passage through a water precipitation bath at 5° C. or roomtemperature membranes were tempered in water for 10 min (Table 2). TABLE2 Membrane formation data. Polymer Temperature Precipi- concentration inof polymer tation Membrane solution [wt-%] solution bath Tempering PAN15 1-2° C. 5° C. 10 min 90° C. AMPS1/1 22.5 RT RT 10 min 65° C. AMPS1/222.5 RT RT 10 min 65° C. AMPS2/1 22.5 RT RT 10 min 65° C.

The formation of membranes from AMPS1/3 with polymer concentration lessthan 15% for getting a larger pore structure was not manageable becausethe material is highly swellable. Hence blend membranes from PAN andAMPS1/3 were formed with varying pore structures. It was assumed thatPAN builds a connected polymer network surrounded by the copolymerAMPS1/3. Data on this membrane formation process are given in Table 3.TABLE 3 Blend membrane formation data. Polymer concentration TemperaturePrecipi- in solution of polymer tation Membrane [wt %] solution bathTempering PAN/AMPS1/3 15 RT RT 10 min 50° C. (1:1) 288 PAN/AMPS1/3 10 RTRT 10 min 50° C. (1:1) 289 PAN/AMPS1/3 10 RT RT 10 min 50° C. (3:1) 290

EXAMPLE 4 Membrane Sieving Characteristics

4.1 Water Permeability and Contact Angles

The membranes formed according to example 3 had water permeabilities andwater contact angles as given in Table 4. Water fluxes of the membraneswere determined in an ultrafiltration device—a Berghoff stirredcell—with distilled water. Water fluxes of AMPS membranes were less thanthose of PAN membranes mainly due to the higher polymer concentrationfor AMPS membrane formation resulting in a more dense structure.Compared to PAN, for the AMPS copolymers significant differences in theadvancing (wetting) and receding (dewetting) water contact angles weremeasured indicating more hydrophilic surfaces of the latter materials.TABLE 4 Water permeabilities and water contact angles of membranes.Water contact angle Water flux advancing receding Membrane [l/m²hkPa][°] [°] PAN 4.02 59.5 43.0 AMPS1/1 0.022 47.3 13.8 AMPS1/2 0.020 54.417.1 AMPS2/1 0.020 30.5 15.6 PAN/AMPS1/3 (1:1) 288 0.200 0 0 PAN/AMPS1/3(1:1) 289 0.838 0 0 PAN/AMPS1/3 (3:1) 290 2.205 0 0

The water fluxes of AMPS blend membranes increase with lower polymerconcentration and higher PAN content in the casting solution. Theseparation parameters of PAN/AMPS1/3 290 are close to the requirementsof a membrane for nutrition supply and waste disposal for tissue cells(water flux about 2 l/m²hkPa) and can be adjusted towards desiredvalues.

For the active surfaces of the blend membranes advancing and recedingwater contact angles of 0° were found indicating highly hydrophilicsurfaces.

4.2 Membrane Performance

To demonstrate the ability to change selectively porosity and transportproperties during membrane formation process, AMPS blend membranes withdifferent polymer concentrations and compositions in casting solutionswere formed according to examples 1 to 3.

Sieving properties of membranes were determined in a Berghoff stirredcell with distilled water and a solution of a test substance mixture(dextrans with molecular weights from 650 to 215.000). Molecular sizedistributions of dextrans in permeates, in retentates and in theoriginal solution were determined using gel permeation chromatography(GPC). From the results of GPC the average pore diameter (D₅₀), themaximal pore diameter (D₁₀₀) and the corresponding molecular weights(cut-off) were calculated by assuming a logarithmic normal distributionof the pore size (Table 5). TABLE 5 Separation ability and performancecharacteristics of PAN/AMPS copolymer blend membranes. Porecharacteristics D₅₀ D₁₀₀ Cut-off Membrane [nm] [nm] σ [kDa] PAN/AMPS1/3(1:1) 288 3.73 7.84 0.2573 23.2 PAN/AMPS1/3 (1:1) 289 8.73 20.08 0.2889188 PAN/AMPS1/3 (3:1) 290 20.75 33.56 0.1668 588

The separation parameters of PAN/AMPS1/3 (3:1) are close to therequirements of a membrane for immunoisolation (cut-off 300 to 400 kDa)and can be changed towards desired values.

Membrane permeability was investigated by measurements of albuminpermeability. A 3 mM albumin solution was used as test solution and PBSas buffer on the sink side of a membrane test cell. Samples were takenevery 20 min from the sink side and were measured for an increase inalbumin concentration by a Lowry assay (Table 6). TABLE 6 Permeabilityfor albumin of PAN/AMPS copolymer blend membranes. Membrane permeabilityMembrane k_(o) [cm/min] PAN/AMPS1/3 (1:1) 288  3.3 × 10⁻⁵ ± 2.8 × 10⁻⁵PAN/AMPS1/3 (1:1) 289 10.5 × 10⁻⁵ ± 7.9 × 10⁻⁵ PAN/AMPS1/3 (3:1) 29010.9 × 10⁻⁵ ± 10.4 × 10⁻⁵

EXAMPLE 5 General Biocompatibility—Contact with 3T3 Fibroblasts

For testing the general biocompatibility of membranes produced accordingto examples 1 to 4, experiments with 3T3 fibroblast were performed. Thecytotoxicity was tested according to IS010993-5 with extracts ofmembranes obtained after 1, 3 and 7 days of incubation of samples incell culture medium (DMEM). As shown by neutral red test, confluentlayers of 3T3 fibroblasts did not show inhibition of viability after 24hours incubation with these extracts indicating no cytotoxic properties(FIG. 1).

Membranes prepared according to examples 1 to 4 were tested forbiocompatibility after 2 and 4 days of direct contact to 3T3fibroblasts. Results were compared to a reference material (Milficell®cell culture insert by Millipore). FIG. 2 shows growth rates of 3T3fibroblasts estimated by the MIT assay. Compared to the referencematerial PAN, copolymer membranes have less favourable surfacesproperties for fibroblasts. The first batch (AMPS1/1) yields stillsignificantly lower growth values for these cells.

EXAMPLE 6 Membranes in Contact with Hepatocytes

6.1. Plating Efficiency and Maintenance of Cytochrome Content

Primary cultures of hepatocytes are renowned for their phenotypicinstability. Moreover, the concentration of total cytochrome P450 andthe activities of its isoenzymes are recognised as the most labilefunctions, extremely sensitive to the culture environment. Thus,hepatocytes were used to assess the maintenance of functions of theprimary cultures on membranes according to examples 1 to 4. If themembranes permitted maintenance of this function it is assumed that theywere likely to be suitable for supporting most of hepatocyte functions.

Hepatocytes were isolated from male Sprague-Dawley rats (180-220 g) bycollagenase perfusion. Viability of 80-90% of isolated cells wasconfirmed by-Trypan Blue exclusion. Hepatocytes were seeded at 1.6×10⁶viable cells per membrane (membrane discs 40 mm in diameter) or 3×10⁶viable cells per collagen-coated (25 μg/cm² Type I collagen isolatedfrom rat tail tendons) 60 mm diameter polystyrene Petri dishes (forcontrol dishes). Cells were cultured in Chee's medium supplemented with5% (v/v) foetal calf serum, in an atmosphere of 5% CO₂ in air. Fourhours after seeding, the cultures were washed to remove unattachedcells, and fresh medium was supplied at this time and once more after 24h. Samples were taken for assessment of the membranes after 48 h.Plating efficiency was determined by measuring total cell protein by theLowry assay. Cytochrome P450 and b5 were measured by a modified methodof Omura and Sato. Results are given in Table 7. TABLE 7 Platingefficiency and cytochrome maintenance of hepatocytes cultured for 48 hon the membranes (control = collagen-coated polystyrene Petri dishes,control cell values = 133.42 pmole/mg protein for cytochrome P450 and155.42 pmole/mg protein for cytochrome b5). Values are means ± SEM,number of experiments given parentheses. Cytochrome maintenance Platingefficiency [% of control] Membrane [%] Cyt P450 Cyt b5 control 66.4 ±16.0 100 100 (6) PAN 63.2 ± 12.0 92.7 ± 43.2 109.2 ± 38.1 (5) (6) (6)AMPS1/1 66.4 ± 16.0 95.3 ± 16.1 107.3 ± 22.8 (5) (5) (5)6.2 Cytochrome P450 Dependent Testosterone Metabolism and O-Deethylationof Ethoxyresorufin (EROD)

The biocompatibility of membranes according to examples 1 to 4 in termsof supporting primary cultures of rat hepatocytes was assessed bymeasuring the cytochrome P450 dependent metabolism of testosterone andethoxyresorufin.

Primary rat hepatocytes isolated according to example 6.1 were seededwith a density of 1.6×10⁶ cells per membrane (cultures on membranediscs, 40 mm in diameter) and 3×10⁶ cells per collagen-coated 60 mmdiameter Petri dish as controls. Cells were cultured in Chee's mediumwith 5% foetal calf serum. After 48 h in culture, cells on the membranesand controls were incubated with 100 μM testosterone in serum-freemedium for 60 min. Products were analysed by HPLC. In a second test, thecells cultured for 48 h were washed, scraped into sodium phosphatebuffer and homogenised using 7 strokes of a motor driven homogeniser.0.5 ml of the homogenates were incubated with 6 μM ethoxyresorufin inthe presence of NAPDH for 10 minutes. Formation of resorufin wasmeasured fluorimetrically.

Table 8 demonstrates a high testosterone metabolism found for rathepatocytes on the AMPS1/1 membrane, although this functionality ofliver cells on the membranes according to the invention is less thanthat on the collagen control. However, the range of hydroxylatedmetabolites formed on the AMPS1/1 membrane was the best of all membranestested. Cells on this membrane formed 6-beta-hydroxy-, 16-alpha-hydroxy,16-beta-hydroxytestosterone and androstenedione, whereas most of theother membranes including the PAN membrane supported only the conversionof testosterone to androstenedione. Table 9 shows that the EROD activityis higher in cells cultured on PAN or AMPS1/1 membranes than oncontrols. TABLE 8 Total testosterone metabolites formed by cell cultures(n = 3) on PAN and AMPS1/1 membranes. Controls = cells cultured oncollagen-coated polystyrene Petri dishes. Total testosterone metabolismMembrane [nmole/h/mg protein] Control 5.4 ± 0.2 PAN 1.5 ± 1.5 AMPS1/13.4 ± 1.5

TABLE 9 The activity of EROD in cells cultured on PAN and AMPS1/1membranes (n = 3). Controls = cells cultured on collagen-coatedpolystyrene Petri dishes. EROD activity [pmole resorufin formed/Membrane min/mg protein] Control 0.6 ± 0.1 PAN 2.0 ± 0.2 AMPS1/1 2.2 ±0.2

The expression of alpha- and pi-isoenzymes of glutathione-S-transferase(GST) changes with de-differentiation and age of liver cells in culture.With culturing time, alpha-GST expression declines whereas that ofpi-GST increases. Expression of both of these GST proteins was detectedin cells growing on the membranes according to the present invention byimmunoblotting using specific antibodies. Table 10 shows expression ofalpha-GST and piGST in cells grown on PAN and AMPS1/1 membranes.Alpha-GSt was well maintained in cells grown on both membranes. Cells onthe AMPS1/1 membrane had particularly low levels of pi-GST suggesting ahigher degree of differentiation. TABLE 10 Expression of alpha-GST andpi-GST in cells cultured on PAN and AMPS1/1 membranes (n = 3). Controlsare cells cultured on collagen-coated Petri dishes. Alpha-GST Pi-GSTMembranes (OD units) (OD units) Control 15.9 ± 0.6  1.5 ± 0.8 PAN 33.7 ±10.4 2.5 ± 1.9 AMPS1/1 24.3 ± 10.0 0.7 ± 0.66.4 Long-Term Performance

At day 7 and 16 of culturing hepatocytes on the AMPS1/1 membrane, cellsfunctions were assessed by the EROD assay, by testosterone metabolismand by GST expression. The effect of collagen coating of the AMPS1/1membrane on the function of the cells was also investigated. As for thePetri dishes control, collagen was coated at 25 μg/cm² on the membrane.The results are shown in Tables 11 and 12 for testosterone metabolism,in Table 13 for the EROD activity and in Table 14 for the GSTs,respectively. Total metabolism of testosterone was less in cellscultured on the membrane than in cells cultured on the Petri dishes atboth 7 and 16 days. Collagen coating of the membrane improved the rangeof metabolites formed by the cells cultured for 7 days on the membrane,but made little difference after 16 days. At day 7, the collagen-coatedmembrane also supported cells which showed lower EROD activities, butagain this effect could not be observed after 16 days. Collagen coatingalso improved the expression of alpha GST in cells cultured on AMPS1/1membrane at 7 days, but not after 16 days of culture. However, there wasalso a high level of expression of pi GST in cells after 7 days cultureon the collagen-coated membrane, but no pi-GST was detectable after 16days with collagen coating.

Long term culture of cells on AMPS1/1 membrane showed that this membranecould maintain viable cells metabolising testosterone andethoxyresorufin and expressing a high level of alpha GST and a low levelof pi GST isoenzymes for 16 days. Collagen coating of the membraneimproved its performance for up to 7 days, but by 16 days collagencoating made little difference to the enzyme activities of the cellscultured on AMPS1/1 membrane. TABLE 11 Total testosterone metabolitesformed by cells cultured for 7 and 16 days on the AMPS1/1 membrane, withand without collagen coating (n = 3). Controls are cells oncollagen-coated Petri dishes. Total testosterone metabolisedMembrane/culture time [nmole/h/mg protein] Control, 7 d 7.2 ± 0.8AMPS1/1, 7 d 1.8 ± 1.0 AMPS1/1 collagen, 7 d 3.7 ± 0.8 Control, 16 d 4.4± 0.8 AMPS1/1, 16 d 2.1 ± 0.4 AMPS1/1 collagen, 16 d 1.6 ± 0.9

TABLE 12 Formation of metabolites from testosterone by cells culturedfor 7 and 16 days on the AMPS1/1 membrane with and without collagencoating (n = 3). Controls are cells on collagen-coated Petri dishes.Values are means ± SEM, the formation of each metabolite is expressed asa percentage of the total metabolites formed (see Table 11). Membrane/6-alpha 6-beta 16-alpha 2-alpha Androstendi culture time Hydroxy HydroxyHydroxy Hydroxy one Control, 7 d 9.3 ± 1.0 29.8 ± 3.1  0 9.5 ± 1.0 51.3± 5.0  AMPS1/1, 7 d 4.6 ± 4.6 30.1 ± 15.4 0 0 32.0 ± 16.0 AMPS1/1 7.5 ±4.0 30.8 ± 2.6  0 3.5 ± 3.5 58.2 ± 6.3  collagen, 7 d Control, 16 d 3.7± 3.7 26.7 ± 1.5  0 6.2 ± 3.1 63.4 ± 4.9  AMPS1/1, 0 6.8 ± 6.8 0 5.1 ±5.1 88.3 ± 6.0  16 d AMPS1/1 0 11.8 ± 11.8 3.5 ± 3.5 0 51.3 ± 28.9collagen, 16 d

TABLE 13 EROD activity of cells cultured for 7 and 16 days on theAMPS1/1 membrane with and without collagen coating (n = 3). Controls arecells cultured on collagen-coated Petri dishes. Values are means ± SEM.EROD activity Membrane/culture time [pmole resorufin formed/min/mgprotein] Control, 7 d 0.8 ± 0.2 AMPS1/1, 7 d 0.8 ± 0.7 AMPS1/1 collagen,7 d 0.4 ± 0.3 Control, 16 d 0.3 ± 0.1 AMPS1/1, 16 d 0.2 ± 0.2 AMPS1/1collagen, 16 d 1.0, 0.9 (n = 2)

TABLE 14 Expression of alpha-GST and pi-GST in cells cultured on AMPS1/1membrane with and without collagen coating for 7 and 16 days (n= 3).Controls are cells cultured on collagen-coated Petri dishes. Values aremeans ± SEM. GST-alpha GST-pi Membrane/culture time [OD units] [ODunits] Control, 7 d 11.2 ± 0.5  0 AMPS1/1, 7 d 11.9 ± 11.1 1 ± 1 AMPS1/1collagen, 7 d 24.0 ± 9.7  11.3 ± 2.6  Control, 16 d 9.7 ± 1.3 4.2 ± 3.7AMPS1/1, 16 d 20.2 ± 3.4  1.6 ± 0.1 AMPS1/1 collagen, 16 d 11.6 ± 5.6  06.5 Morphology

Hepatocytes isolated and cultured according to example 6.1 were seededon AMPS1/1 membranes according to examples 1 to 4 and incubated up to 20days. Some of the membranes were coated with collagen before seeding thecells. As control, hepatocytes were also cultured on collagen-coatedPetri dishes. The morphology of the hepatocytes on the AMPS1/1 membraneswith and without collagen coating was compared with the control. Cellviability was assessed at day 7, 16 and 20 of culture usingcarboxyfluorescein diacetate (CFDA) and confocal microscopy. Viablecells were maintained at all time points. The membrane according to thepresent invention supported cell viability as well as thecollagen-coated Petri dishes did. On AMPS1/1 membrane cells showed goodmorphology, with highly developed bile cannaliculi.

1-30. (canceled)
 31. A method for enhancing the biocompatibility of amaterial for use in medical or biological applications involving adirect contact of the material with at least one of fluids, cells andtissues, comprising the step of producing the material from a polymericcomposition comprising at least one acrylic copolymer, said acryliccopolymer consisting of: a) 90 to 99 mole-% of acrylonitrile (AN)monomer units; and b) 1 to 10 mole-% of 2-acrylamido-2-methyl-propanesulfonic acid (AMPS) as a water-soluble comonomer unit.
 32. The methodaccording to claim 31, where the 2-acrylamido-2-methyl-propane sulfonicacid (AMPS) is ionic.
 33. The method according to claim 31, wherein thecopolymer comprises 3 to 5 mole-% of 2-acrylamido-2-methylpropanesulfonic acid (AMPS).
 34. The method according to claim 31, wherein thematerial is in the form of a membrane, a film or a surface coating. 35.The method according to claim 34, wherein the membrane, the film or thecoating comprises a blend of said polymeric composition andpoly-acrylonitrile (PAN).
 36. The method according to claim 31, whereinthe material is part of a biohybrid or bioartificial organ comprisingtissue cells immobilized on said material.
 37. The method according toclaim 36, wherein the tissue cells are selected from liver cells,pancreas cells and lung cells.
 38. The method according to claim 37,wherein the liver cells are hepatocytes.
 39. The method according toclaim 36, wherein the material is in form of a membrane separating thetissue cells immobilized on the membrane from a fluid stream, whichsupplies the cells with nutrients and enables exchange of substancesinto the cells and outside the cells.
 40. The method according to claim34, wherein the membrane is an asymmetric membrane, comprising an outerdense layer having an average pore size of 1 to 50 nm.
 41. The methodaccording to claim 34, wherein the membrane has a rate of water fluxthrough the membrane in the range of 1 to 10 l/m²hkPa.
 42. The methodaccording to claim 34, wherein the membrane has a cut-off in the rangeof 150 to 1,000 kDa.
 43. The method according to claim 34, wherein themembrane is formed by a phase-inversion process.
 44. The methodaccording to claim 31, wherein the material is an implant or biosensorfor medical applications within the body.
 45. The method according toclaim 31, wherein the material is in form of a flat or hollow fibremembrane having at least a two-layer cross sectional structuresubstantially consisting of a dense surface layer and a porous bulklayer having finger-like pores communicating with the dense layer.